Comparative Analysis of Active Ingredients and Potential Bioactivities of Essential Oils from Artemisia argyi and A. verlotorum

Artemisia argyi H. Lév. and Vaniot is a variety of Chinese mugwort widely cultured in central China. A. verlotorum Lamotte, another variety of Chinese mugwort, has been used in the southern region of China since ancient times. Despite their similar uses in traditional medicine, little is known about the differences in their active ingredients and potential benefits. Herein, the chemical compositions of the essential oils (EOs) from both varieties were analyzed using chromatography-mass spectrometry (GC-MS). A series of databases, such as the Traditional Chinese Medicine Systems Pharmacology database (TCMSP), SuperPred database and R tool, were applied to build a networking of the EOs. Our results revealed significant differences in the chemical compositions of the two Artemisia EOs. However, we found that they shared similar ingredient–target–pathway networking with diverse bioactivities, such as neuroprotective, anti-cancer and anti-inflammatory. Furthermore, our protein connection networking analysis showed that transcription factor p65 (RELA), phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1) and mitogen-activated protein kinase 1 (MAPK1) are crucial for the biological activity of Artemisia EOs. Our findings provided evidence for the use of A. verlotorum as Chinese mugwort in southern China.


Introduction
Artemisia spps. are grown in Asia, North America and Europe [1]. They are widely used as aromatic ethnic medicines in China, with their essential oils (EOs) being the major active ingredients frequently used in pharmaceuticals and cosmetics [2]. Due to the synergistic effects of a number of their active components, Artemisia EOs have pleiotropic bioactivity [3] and have attracted considerable attention for their biological diversities and bioactivities.

Comparison and Analysis of the Chemical Composition of AAEOs and AVEOs
The chemical compositions of the Vs were more abundant than the AVEOs and differed significantly. Among them, neointermedeol and eucalyptol were the main ingredients of the AAEOs (Figure 2), while caryophyllene oxide was the primary component of the AVEOs (Figure 3). Furthermore, the content of monoterpenoids from the AAEOs (>30%) was higher than that of the AVEOs (<5%). However, sesquiterpenes had a higher proportion in the AVEOs (>60%) than in the AAEOs (<52%). Moreover, diterpenoids were only found in the whole grass of both AAEOs and AVEOs. The contents of monoterpenoids and sesquiterpenes may be used as markers to distinguish AAEOs from AVEOs.

Comparison and Analysis of the Chemical Composition of AAEOs and AVEOs
The chemical compositions of the Vs were more abundant than the AVEOs and differed significantly. Among them, neointermedeol and eucalyptol were the main ingredients of the AAEOs (Figure 2), while caryophyllene oxide was the primary component of the AVEOs (Figure 3). Furthermore, the content of monoterpenoids from the AAEOs (>30%) was higher than that of the AVEOs (<5%). However, sesquiterpenes had a higher proportion in the AVEOs (>60%) than in the AAEOs (<52%). Moreover, diterpenoids were only found in the whole grass of both AAEOs and AVEOs. The contents of monoterpenoids and sesquiterpenes may be used as markers to distinguish AAEOs from AVEOs.

Analysis of the Main Active Ingredients and the Potential Effect of EOs from A. Argyi and A. Verlotorum Using the Ingredient-Target-Pathway Networking
We analyzed the active ingredients, key targets and pathways of the different Artemisia EOs and created an "ingredient-target-pathway" map with a merge function. The networking was visualized using Cytoscape 3.9.1 software ( Figure S2). The ingredients were represented by the purple V, the targets were represented by the yellow ellipse and the pathways were represented by the blue rectangle. The active ingredients were represented by the node, and the edge links represented the targets and the active ingredients. The high number of linkages demonstrated the importance of networking the active The whole grasses of A. argyi were more chemically diverse than the leaves. The main chemical substances of the EOs from the two parts of A. argyi were similar, while the contents had some differences. There were 42 identical components, including neointermedeol, caryophyllene, caryophyllene oxide, etc., (Table S2) in both parts of the EOs. Neointermedeol and caryophyllene were the principal components of the whole grass (9.69% and 8.73%, respectively). They were also present at a high content in the leaves (8.02% and 9.24%, respectively). The main constituents of the LAAEOs were eucalyptol (11.51%) and 2-borneol (10.09%). However, the content of these two ingredients in the GAAEOs was only 5.77% and 4.54%, respectively. Notably, the LAAEOs had fewer compounds, but a higher content of major components than the GAAEOs, which may be one of the reasons why the leaves have been commonly used as medicinal herbs.

Comparison and Analysis of the EOs from Whole grass and Leaves of A. verlotorum
The A. verlotorum used in this study was collected in Guangdong province, China. For the whole grass of A. verlotorum (GAVEOs, YP-3) ( Figure 3A, Table S1), we detected 47 ingredients, including two monoterpenoids (1.05%), 37 sesquiterpenoids (63.56%), two diterpenoids, one aromatic compounds (3.36%), four fatty compounds (6.68%) and one phosphate (0.42%). Among them, caryophyllene oxide (17.06%), (+)-β-eudesmol (6.67%), neointermedeol (4.72%), α-gurjunene (3.82%) and isospathulenol (3.77%) were the major sesquiterpenoids. Similarly to the LAVEOs, the GAVEOs had a higher content and diverse structure of sesquiterpenes compared to monoterpenes. It is clear that the yield and constitutes of Artemisia oils may be influenced by the cultivation location, harvest time, vegetative cycle stage, extraction method and selection of plant parts, based on preliminary studies [18,28]. A comparison of the variance of terpenoid biosynthesis among different parts of Artemisia from the gene perspective suggested that there was a significant difference in the expression pattern of genes [29].
The chemical composition of the GAVEOs was more abundant than that of the LAVEOs. Diterpenoids and aromatics were detected only in the whole grass. Interestingly, caryophyllene oxide (>17%) was the main substance in the EOs from different parts of A. verlotorum. Thirty common constituents were identified in the LAVEOs and GAVEOs, such as himbaccol, (+)-β-eudesmol, isospathulenol, etc. (Table S2).

Analysis of the Main Active Ingredients and the Potential Effect of EOs from A. argyi and A. verlotorum Using the Ingredient-Target-Pathway Networking
We analyzed the active ingredients, key targets and pathways of the different Artemisia EOs and created an "ingredient-target-pathway" map with a merge function. The networking was visualized using Cytoscape 3.9.1 software ( Figure S2). The ingredients were represented by the purple V, the targets were represented by the yellow ellipse and the pathways were represented by the blue rectangle. The active ingredients were represented by the node, and the edge links represented the targets and the active ingredients. The results showed that the ten components with the highest connectivity in these EOs differed. In contrast, the targets of those ten components were quite similar. Moreover, they corresponded to exactly the same pathways ( Figure 4, Table S3). The results suggest that although the components are different, they have similar biological activities.
networking edges. The nodes of the LAAEOs (YP-2) included 54 components, 329 targets and 287 pathways, where trimethylenenorbornane had the highest number of networking edges. The nodes of the GAVEOs (YP-3) included 39 components, 278 targets and 275 pathways, where pentamethylcyclopentadiene had the highest number of networking edges. The nodes of the LAVEOs (YP-4) included 50 components, 284 targets and 280 pathways, where 9-(1-methylethylidene)-1,5-cycloundecadiene had the highest number of networking edges. The results showed that the ten components with the highest connectivity in these EOs differed. In contrast, the targets of those ten components were quite similar. Moreover, they corresponded to exactly the same pathways ( Figure 4, Table S3). The results suggest that although the components are different, they have similar biological activities.

Comparison and Analysis of Main Active Ingredients of AAEOs and AVEOs
In order to focus on the possible active components, we investigated the drug-like characteristics of these molecules. The discovered compounds in the AAEOs and AVEOs were evaluated for their drug-like characteristics using Lipinski's five guidelines. R was applied to analyze the correlation between the nodes. The nodes with a strong correlation were displayed with the same/similar color ( Figure S3). The active ingredients contained in the top 50 scores are shown in Table 2, and the results showed that the ingredients with the highest scores among the four EOs were not the same.

Comparison and Analysis of Main Active Ingredients of AAEOs and AVEOs
In order to focus on the possible active components, we investigated the drug-like characteristics of these molecules. The discovered compounds in the AAEOs and AVEOs were evaluated for their drug-like characteristics using Lipinski's five guidelines. R was applied to analyze the correlation between the nodes. The nodes with a strong correlation were displayed with the same/similar color ( Figure S3). The active ingredients contained in the top 50 scores are shown in Table 2, and the results showed that the ingredients with the highest scores among the four EOs were not the same.
Methyleugenol, the highest scoring compound in the GAAEOs, was a phenylpropanoid used as a flavoring agent, a fragrance and an anesthetic in rodents [32,33]. However, the content of methyleugenol was only 0.17% in the GAAEOs, and it is unclear whether it is related to the activity of the EOs. In addition, no clear bioactivity studies have been reported for alloaromadendrene oxide (the highest-scoring compound in LAAEOs), m-anisalcohol (the highest-scoring compound in GAVEOs) and 9-(1-methylethylidene)-1,5-cycloundecadiene (the highest-scoring compound in LAVEOs).
The caryophyllene oxide and neointermedeol were the common components of these four Eos, as shown in Table 2 using Venny analysis ( Figure 5). Caryophyllene oxide has broad activities that have attracted much attention. It exhibited cytotoxicity against multiple cancer cells, including MG-63 (human osteosarcoma cells), HepG2 (human leukemia cancer cells), AGS (human lung cancer cells) and SNU-1 (human gastric cancer cell) [34,35]. Another study showed that caryophyllene oxide is a potent CNS depressant [36]. In addition, the CYP3A enzyme activity was markedly decreased by caryophyllene oxide, which could generally have an impact on the pharmacokinetics of the active compounds [37]. However, few studies have reported the biological activity of neointermedeol. a Numbering of compounds in the R cluster analysis diagram ( Figure S3). b Scores of components from R cluster analysis.
Molecules 2022, 27, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molecules broad activities that have attracted much attention. It exhibited cytotoxicity against multiple cancer cells, including MG-63 (human osteosarcoma cells), HepG2 (human leukemia cancer cells), AGS (human lung cancer cells) and SNU-1 (human gastric cancer cell) [34,35]. Another study showed that caryophyllene oxide is a potent CNS depressant [36]. In addition, the CYP3A enzyme activity was markedly decreased by caryophyllene oxide, which could generally have an impact on the pharmacokinetics of the active compounds [37]. However, few studies have reported the biological activity of neointermedeol.  Table 2. Venny analysis found that the EOs from different parts of two Artemisia share the same ingredients of caryophyllene oxide and neointermedeol. (B) Chemical structures of caryophyllene oxide and neointermedeol.

Comparison and Analysis of Key Proteins of AAEOs and AVEOs
Compounds show bioactivities by binding with particular proteins. [38]. Table 3 shows the target proteins included in the top 50 scores after clustering analysis by R. Among them, NFKB1 has the highest score in all of the essential oils. NFKB1 is a member of the NF-κB family and an important regulator of NF-κB activity in vivo. It has been shown that NFKB1 is closely associated with inflammation, aging and cancer in the body [39][40][41][42]. Thus, NFKB1 is one of the key targets of Artemisia essential oils with similar biological activity.
A protein association network was constructed using STRING databases to screen the key target proteins with high interactions (Figure 6). The nodes encoded the networking of the target proteins. Furthermore, the protein-protein connection was defined as the connectivity degree. Genes with a high connectivity degree were defined as hub genes. The study found that RELA (transcription factor p65), PIK3R1 (phosphatidylinositol 3kinase regulatory subunit α) and MAPK1 (mitogen-activated protein kinase 1) were the key targets and played a crucial role in various functions of the EOs from Artemisia in  Table 2. Venny analysis found that the EOs from different parts of two Artemisia share the same ingredients of caryophyllene oxide and neointermedeol. (B) Chemical structures of caryophyllene oxide and neointermedeol.

Comparison and Analysis of Key Proteins of AAEOs and AVEOs
Compounds show bioactivities by binding with particular proteins. [38]. Table 3 shows the target proteins included in the top 50 scores after clustering analysis by R. Among them, NFKB1 has the highest score in all of the essential oils. NFKB1 is a member of the NF-κB family and an important regulator of NF-κB activity in vivo. It has been shown that NFKB1 is closely associated with inflammation, aging and cancer in the body [39][40][41][42]. Thus, NFKB1 is one of the key targets of Artemisia essential oils with similar biological activity.  Figure S3). d Scores of targets from R cluster analysis.
A protein association network was constructed using STRING databases to screen the key target proteins with high interactions (Figure 6). The nodes encoded the net-working of the target proteins. Furthermore, the protein-protein connection was defined as the connectivity degree. Genes with a high connectivity degree were defined as hub genes. The study found that RELA (transcription factor p65), PIK3R1 (phosphatidylinositol 3-kinase regulatory subunit α) and MAPK1 (mitogen-activated protein kinase 1) were the key targets and played a crucial role in various functions of the EOs from Artemisia in therapy. RELA (RELA Proto-Oncogene, NF-κB Subunit) is a pleiotropic transcription factor in practically all cell types. It is the endpoint of a series of signal transduction events that are sparked by various stimuli related to numerous biological processes, including tumorigenesis, differentiation, cell growth and apoptosis [43][44][45][46]. PIK3R1 functions as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane. It binds to activated (phosphorylated) protein-Tyr kinases through its SH2 domain. This is required for the insulin-stimulated increase in glucose uptake and glycogen synthesis in insulin-sensitive tissues [47,48]. These targets are of great significance and deserve further investigation based on the relevant biological activity research before AAEOs, including their anti-inflammatory [9,49], neuroprotection [50], hypoglycemic and anti-oxidant [51], as well as anti-cancer [52,53] and other beneficial pharmacological, effects [18,54].

GO Enrichment Analysis and KEGG Pathway Annotation
The biological processes, cellular components and molecular functions were the three major functional categories identified from the GO term enrichment analysis of the EOs. The top ten GO terms of each category are illustrated in Figure S4. The results showed that the cellular components had the most significantly enriched terms, with protein binding being the most significant cellular function among the four EOs. Protein binding can improve or impair a drug's effectiveness [57].
To identify the pathways and targets that were involved directly, the pathway information related to the targets was obtained through KEGG analysis. Figure 7 shows that the associated pathways mainly included metabolic pathways, the neuroactive ligandreceptor interaction and pathways in cancer. Substantial experimental evidence showed that AAEOs had good activities in neuroprotection [50], anti-inflammatory, analgesic and anti-tumor effects [18,54], which were consistent with the prediction of networking pharmacology. Further analysis based on GO and KEGG showed that the enrichment results of the AAEOs and AVEOs were highly consistent, indicating that AVEOs and AAEOs Additionally, the study found that the two MAPKs that are crucial to the MAPK/ERK cascade are MAPK1/ERK2 and MAPK3/ERK1. The regulation of transcription, translation and cytoskeletal rearrangements by the MAPK/ERK cascade regulates a variety of biological tasks, including cell growth, adhesion, survival and differentiation, depending on the cellular context [55,56]. Our results showed that although the ingredients were different from AVEOs and AAEOs, the targets of those ingredients were the same proteins. The results suggested that AVEOs and AAEOs have similar biological activities.

GO Enrichment Analysis and KEGG Pathway Annotation
The biological processes, cellular components and molecular functions were the three major functional categories identified from the GO term enrichment analysis of the EOs. The top ten GO terms of each category are illustrated in Figure S4. The results showed that the cellular components had the most significantly enriched terms, with protein binding being the most significant cellular function among the four EOs. Protein binding can improve or impair a drug's effectiveness [57].
To identify the pathways and targets that were involved directly, the pathway information related to the targets was obtained through KEGG analysis. Figure 7 shows that the associated pathways mainly included metabolic pathways, the neuroactive ligand-receptor interaction and pathways in cancer. Substantial experimental evidence showed that AAEOs had good activities in neuroprotection [50], anti-inflammatory, analgesic and anti-tumor effects [18,54], which were consistent with the prediction of networking pharmacology. Further analysis based on GO and KEGG showed that the enrichment results of the AAEOs and AVEOs were highly consistent, indicating that AVEOs and AAEOs might have the same action pathway and similar pharmacological activities.

Network Analysis of the Unique Components of the Four Artemisia Essential Oils
Furthermore, we constructed a network between the unique composition of Artemisia essential oils (GAAEOs, LAAEOs, GAVEOs, LAVEOs) and its targets and pathways (Figure 8). The key targets and pathways of the top five (red nodes) revealed by the enrichment results were highly consistent with the results of the GO and KEGG analyses ( Figure  6 and Figure 7), further illustrating that the two different Artemisia essential oils have similar pharmacological activities despite significantly differing in their compositions. In addition, the key targets have been proved to mediate the relevant signaling pathways to exert immunomodulatory, anti-inflammatory and neuroprotective activities, etc. [18,58-

Network Analysis of the Unique Components of the Four Artemisia Essential Oils
Furthermore, we constructed a network between the unique composition of Artemisia essential oils (GAAEOs, LAAEOs, GAVEOs, LAVEOs) and its targets and pathways ( Figure 8). The key targets and pathways of the top five (red nodes) revealed by the enrichment results were highly consistent with the results of the GO and KEGG analyses (Figures 6 and 7), further illustrating that the two different Artemisia essential oils have similar pharmacological activities despite significantly differing in their compositions. In addition, the key targets have been proved to mediate the relevant signaling pathways to exert immunomodulatory, anti-inflammatory and neuroprotective activities, etc. [18,[58][59][60].

In Vitro and In Vivo Toxicity of Artemisia Essential Oils
As a processing of Chinese materia medica widely used in clinic, the toxicological evaluation of the Artemisia essential oils was particularly important. In order to better ensure the drug safety, it can be assumed that the biological activity of Artemisia essential oils was assessed through in vitro and in vivo toxicity tests. The results of the cellular level assay showed that 100 μg/mL of Artemisia essential oils did not show cytotoxic activity against both HEK-293T cell lines treated for 24 h (Figure 9, A and B). Furthermore, the in vivo assay in zebrafish showed that 10 μg/mL of Artemisia essential oils did not affect the growth and survival of zebrafish (Figure 9, C and D). This study presents the first systematic assessment of the toxicity of Artemisia essential oils using an in vitro human normal cell line (HEK-293T cells) assay in combination with an in vivo zebrafish assay. In this regard, Artemisia essential oils are highly safe.

Conclusions
Aromatic chemical components from folkloric medicinal plants, such as EOs, have been claimed to be useful in treating or preventing a variety of illnesses [61]. The fragrant medicinal plants of the Artemisia species have complicated locations and origins [1]. Although the compounds and activity of A. argyi have been investigated, the mechanism by which these components act on human health at the cell level has remained largely unknown. Few studies have focused on the biological activity of the essential oils of A. verlotorum.
Herein, we analyzed the compositional differences and potential bioactivities of EOs from two Chinese mugworts. The results showed that the chemical composition of the EOs from A. argyi and A. verlotorum were quite different. However, networking pharmacology and R cluster analysis studies showed that they share similar key target proteins, as well as highly consistent protein interactions and signaling pathways. This indicated that these two Artemisia essential oils could be substituted for each other in aromatic therapy. Our study provides evidence to better understand the development and application of A. argyi and A. verlotorum in Chinese traditional medicine and lays a foundation for the clinical safety and scientific medication of Artemisia essential oils.

In Vitro and In Vivo Toxicity of Artemisia Essential Oils
As a processing of Chinese materia medica widely used in clinic, the toxicological evaluation of the Artemisia essential oils was particularly important. In order to better ensure the drug safety, it can be assumed that the biological activity of Artemisia essential oils was assessed through in vitro and in vivo toxicity tests. The results of the cellular level assay showed that 100 µg/mL of Artemisia essential oils did not show cytotoxic activity against both HEK-293T cell lines treated for 24 h ( Figure 9A,B). Furthermore, the in vivo assay in zebrafish showed that 10 µg/mL of Artemisia essential oils did not affect the growth and survival of zebrafish ( Figure 9C). This study presents the first systematic assessment of the toxicity of Artemisia essential oils using an in vitro human normal cell line (HEK-293T cells) assay in combination with an in vivo zebrafish assay. In this regard, Artemisia essential oils are highly safe.

Plant Materials and Reagent
The A. argyi H. Lév. and Vaniot and A. verlotorum Lamotte were derived from different species of the same genus and have extremely similar morphological characteristics. These samples were (n = 3) collected from Nanyang, Henan Province, and Luofo mountain, Guangdong Province (specific medicinal plant cultivation sites) [62,63], People's Republic of China, respectively, and were collected strictly according to the medicinal age (1 year) and time of harvest. Both plants were identified by Prof. Chong-Ren Yang. The materials were dried in the shade at 25 °C until the humidity was lower than 5%, and stored in the refrigerator at 4 °C. Voucher specimens (more than 100 mg) were deposited at the Center for Pharmaceutical Sciences, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, P. R. China.

Extraction of EOs A. Verlotorum
Steam distillation was used to extract the EOs from different parts of the whole grass and leaves of A. argyi (YP-1 and YP-2) and A. verlotorum (YP-3 and YP-4). The dried Artemisia (100 g), in a round bottom flask, was added to distilled water (1 L). The extractions of Eos, in detail, followed previous studies [64]. Detailed information of all the EOs is presented in Table 1.

Conclusions
Aromatic chemical components from folkloric medicinal plants, such as EOs, have been claimed to be useful in treating or preventing a variety of illnesses [61]. The fragrant medicinal plants of the Artemisia species have complicated locations and origins [1]. Although the compounds and activity of A. argyi have been investigated, the mechanism by which these components act on human health at the cell level has remained largely unknown. Few studies have focused on the biological activity of the essential oils of A. verlotorum.
Herein, we analyzed the compositional differences and potential bioactivities of EOs from two Chinese mugworts. The results showed that the chemical composition of the EOs from A. argyi and A. verlotorum were quite different. However, networking pharmacology and R cluster analysis studies showed that they share similar key target proteins, as well as highly consistent protein interactions and signaling pathways. This indicated that these two Artemisia essential oils could be substituted for each other in aromatic therapy. Our study provides evidence to better understand the development and application of A. argyi and A. verlotorum in Chinese traditional medicine and lays a foundation for the clinical safety and scientific medication of Artemisia essential oils.

Plant Materials and Reagent
The A. argyi H. Lév. and Vaniot and A. verlotorum Lamotte were derived from different species of the same genus and have extremely similar morphological characteristics. These samples were (n = 3) collected from Nanyang, Henan Province, and Luofo mountain, Guangdong Province (specific medicinal plant cultivation sites) [62,63], People's Republic of China, respectively, and were collected strictly according to the medicinal age (1 year) and time of harvest. Both plants were identified by Prof. Chong-Ren Yang. The materials were dried in the shade at 25 • C until the humidity was lower than 5%, and stored in the refrigerator at 4 • C. Voucher specimens (more than 100 mg) were deposited at the Center for Pharmaceutical Sciences, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, P. R. China.

Extraction of EOs A. verlotorum
Steam distillation was used to extract the EOs from different parts of the whole grass and leaves of A. argyi (YP-1 and YP-2) and A. verlotorum (YP-3 and YP-4). The dried Artemisia (100 g), in a round bottom flask, was added to distilled water (1 L). The extractions of Eos, in detail, followed previous studies [64]. Detailed information of all the EOs is presented in Table 1.

GC-MS Analysis
GC-MS was used to analyze the compounds of AAEOs and AVEOs. The methods, in detail, followed previous studies [64].
Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http: //www.kegg.jp/kegg/, 4 March 2022) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/summary.jsp, 4 March 2022), the analysis of pathways was carried out on the chosen targets. The database is an encyclopedia of genes and genomes and includes information on signal transduction, cellular biology and homologous conservative route [68]. The study uses Human sapiens as model species.

Networking Construction
Cytoscape 3.9.1 was used to analyze and visualize the data gathered to create complicated networks [69]. In this network, nodes stood in for components, targets or pathways, while edges denoted their connections. Subsequently, the tight lines and complexities of the connections between important chemical components, targets and pathways were considered to explore the underlying mechanism of action. Therefore, cluster analysis of the relevant collected information was performed by R (cluster_louvain). The related ingredients, targets and pathways information resulted in a data set that was converted to an igraph graph using the "igraph" software package. A function of graph_from_incidence_matrix creates a bipartite igraph graph from the incidence matrix of the data for targets and pathways. Based on the bipartite igraph graph, a function of igraph_cluster_louvain implements the multi-level modularity optimization algorithm for finding community structure, and different communities were marked in different colors by a R_rainbow, while their relationships were analyzed using the igraph_deg function [70].
Based on the top targets in the R processing results, protein-protein interaction networking analysis (PPI) was also carried out to assess the targets. The association between the targets was evaluated as follows. PPI: To show how the target proteins interact, the target proteins were uploaded to the STRING databases platform (http://string-db.org, 6 April 2022). In this study, we eliminated the isolated targets and constructed a PPI networking by screening them with a confidence score > 0.90.

Gene Ontology and Pathway Enrichment Analysis
Gene ontology (GO) enrichment analysis was performed on the candidate targets using the online tool DAVID and KEGG, as well as KEGG pathway annotation. p values < 0.05 were considered statistically significant.

Toxicity Analysis
Cytotoxicity assay: The toxicity of Artemisia essential oils on HEK-293T cells (human embryonic kidney 293 cells, were obtained from the Kunming Cell Bank of Chinese Academic of Sciences) was detected using the MTT method, consistent with the previous study [71]. Cells were treated with Artemisia essential oils (25, 50, 100 µg/mL) for 24 h. The cells incubated with 3 µM paclitaxel (PTX) (Adamas Reagent Co., Ltd., 48803A, Shanghai, China) was used as the positive control (paclitaxel was drugs widely used clinically for the treatment of cancer).
Zebrafish toxicity assay: Healthy 24 h post-fertilization (hpf) embryos (transparent fertilized eggs) were randomly transferred to different concentrations of methanol extract of Artemisia essential oils (10 µg/mL) in the sterile 12 well plate. Each group was provided with 20 embryos (repeated experiment, n = 5). The development and morphological changes of the embryos after drug exposure were observed, photographed and recorded using an inverted optical microscope at 18 and 36 hpf, and recorded the hatching rate of the embryos at 36 hpf.

Statistical Analysis
All the data were statistically analyzed using the GraphPad Prism 9 software, using a two-tailed Student's t-test. p values < 0.05 were considered statistically significant.